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Although several human clinical trials using various bone marrow-derived cell types for cirrhotic or decompensated patients have reported a short-term benefit, long-term follow-up data are limited. We analyzed the long-term clinical outcomes of autologous bone marrow cell infusion (ABMI) for decompensated liver cirrhosis (LC). Patients enrolled in a pilot single-armed ABMI study were followed up more than 5 years. Bone marrow-derived mononuclear cells (BM-MNCs) from decompensated LC were harvested and after processing were infused into a peripheral vein. The laboratory test results and long-term clinical course including liver transplantation (LT), development of cancer, cause of death, and survival after ABMI were analyzed. Nineteen patients were followed up for a median of 66 months after ABMI. Liver function, including serum levels of albumin and Child–Pugh (CP) score, was improved at the 1-year follow-up. Liver volume was significantly greater, cirrhosis was sustained, and collagen content was decreased at the 6-month follow-up. Five years after ABMI, five patients (26.3%) maintained CP class A without LT or death, and five patients (26.3%) had undergone elective LT. Hepatocellular carcinoma (HCC) occurred in five patients (26.3%), and lymphoma and colon cancer occurred in one patient each. Three patients (15.8%) were lost to follow-up at months 22, 31, and 33, respectively, but maintained CP class A until their last follow-up. Five patients expired due to infection. While improved liver function was maintained in some patients for more than 5 years after ABMI, other patients developed HCC. Further studies of long-term follow-up cohorts after cell therapy for LC are warranted.

Chromatin organization is essential for appropriate interpretation of the genetic information. Here, we demonstrated that the chromatin-associated proteins HP1 are dispensable for hepatocytes survival but are essential within hepatocytes to prevent liver tumor development in mice with HP1β being pivotal in these functions. Yet, we found that the loss of HP1 per se is not sufficient to induce cell transformation but renders cells more resistant to specific stress such as the expression of oncogenes and thus in fine, more prone to cell transformation. Molecular characterization of HP1-Triple KO premalignant livers and BMEL cells revealed that HP1 are essential for the maintenance of heterochromatin organization and for the regulation of specific genes with most of them having well characterized functions in liver functions and homeostasis. We further showed that some specific retrotransposons get reactivated upon loss of HP1, correlating with overexpression of genes in their neighborhood. Interestingly, we found that, although HP1-dependent genes are characterized by enrichment H3K9me3, this mark does not require HP1 for its maintenance and is not sufficient to maintain gene repression in absence of HP1. Finally, we demonstrated that the loss of TRIM28 association with HP1 recapitulated several phenotypes induced by the loss of HP1 including the reactivation of some retrotransposons and the increased incidence of liver cancer development. Altogether, our findings indicate that HP1 proteins act as guardians of liver homeostasis to prevent tumor development by modulating multiple chromatin-associated events within both the heterochromatic and euchromatic compartments, partly through regulation of the corepressor TRIM28 activity.

To determine the origin of adult tissue-resident macrophages, a mouse lineage tracing study has revealed that these cells derive from erythro-myeloid progenitors in the yolk sac that are distinct from fetal and adult haematopoietic stem cells. The origin of adult myeloid cells The developmental origin of tissue-resident macrophage progenitors and their contribution to macrophages in fetal and adult organs relative to bone marrow macrophages are still unclear. Using lineage tracing, Elisa Gomez Perdiguero et al . identify a population of yolk-sac-derived progenitors — distinct from fetal and adult haematopoetic stem cells — that gives rise to erythrocytes, macrophages, granulocytes and monocytes in the young mouse fetus, and to the vast majority of adult tissue-resident macrophages. Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs) 1 , 2 , 3 , however, macrophages in adult tissues can self-maintain independently of HSCs 4 , 5 , 6 , 7 . Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs 8 , 9 , 10 , 11 , 12 , 13 , and fetal macrophages 13 , 14 , 15 can develop independently of Myb 4 , a transcription factor required for HSC 16 , and can persist in adult tissues 4 , 17 , 18 . Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear 19 . Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2 + (also known as Tek ) cellular pathway generating Csf1r + erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages.

Cancer is a clonal disorder derived from a single ancestor cell and its progenies that are positively selected by acquisition of ‘driver mutations’. However, the evolution of positively selected clones does not necessarily imply the presence of cancer. On the contrary, it has become clear that expansion of these clones in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation. Recent studies have reported expansion of clones harbouring mutations in cancer driver genes in the blood, skin, oesophagus, bronchus, liver, endometrium and bladder, where the expansion could be so extensive that tissues undergo remodelling of an almost entire tissue. The presence of common cancer driver mutations in normal tissues suggests a strong link to cancer development, providing an opportunity to understand early carcinogenic processes. Nevertheless, some driver mutations are unique to normal tissues or have a mutation frequency that is much higher in normal tissue than in cancer, indicating that the respective clones may not necessarily be destined for evolution to cancer but even negatively selected for carcinogenesis depending on the mutated gene. Moreover, tissues that are remodelled by genetically altered clones might define functionalities of aged tissues or modified inflammatory processes. In this Review, we provide an overview of major findings on clonal expansion in phenotypically normal or non-cancer tissues and discuss their biological significance not only in cancer development but also in ageing and inflammatory diseases.Clonal expansion in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation, but does not necessarily indicate cancer development. This Review discusses recent findings on clonal expansion in these tissues and their biological significance in cancer development, ageing and inflammatory diseases.

The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment. The development of single cell RNA sequencing technologies has been instrumental in advancing our understanding of tissue biology. Here, MacParland et al. performed single cell RNA sequencing of human liver samples, and identify distinct populations of intrahepatic macrophages that may play specific roles in liver disease.

Key Points Liver macrophages comprise Kupffer cells — which are self-maintaining, non-migratory tissue-resident phagocytes that originate from yolk sac-derived precursors during embryogenesis — and monocyte-derived macrophages. Kupffer cells are essential for hepatic and systemic homeostasis, as they contribute to metabolism, scavenge bacteria and cellular debris, and induce immunological tolerance. Following their activation by danger signals, Kupffer cells modulate inflammation and recruit immune cells — including large numbers of monocytes — to the liver. Kupffer cells and monocyte-derived macrophages rapidly adapt their phenotypes in response to local signals, which determine their ability to aggravate or cease liver injury. Liver macrophages are crucial in the pathogenesis of acute and chronic liver diseases, in which they orchestrate inflammation, fibrosis, angiogenesis and tumour progression, as well as tissue repair and tumour surveillance. Evidence from animal models and early clinical trials in humans indicates that targeting pathogenic liver macrophages might be a promising therapeutic approach in acute and chronic liver diseases. This Review describes the different populations of monocytes and macrophages, including Kupffer cells, that are found in the liver. The authors discuss the immune functions of these cells in the homeostatic liver as well as during liver infection and disease. Macrophages represent a key cellular component of the liver, and are essential for maintaining tissue homeostasis and ensuring rapid responses to hepatic injury. Our understanding of liver macrophages has been revolutionized by the delineation of heterogeneous subsets of these cells. Kupffer cells are a self-sustaining, liver-resident population of macrophages and can be distinguished from the monocyte-derived macrophages that rapidly accumulate in the injured liver. Specific environmental signals further determine the polarization and function of hepatic macrophages. These cells promote the restoration of tissue integrity following liver injury or infection, but they can also contribute to the progression of liver diseases, including hepatitis, fibrosis and cancer. In this Review, we highlight novel findings regarding the origin, classification and function of hepatic macrophages, and we discuss their divergent roles in the healthy and diseased liver.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs) 1 . However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45 + haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45 + CD34 + CD44 + yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs. Single-cell RNA sequencing of haematopoietic cells from human embryos at different developmental stages sheds light on the development and specification of macrophages in different tissues.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes 1 – 7 . Stem cells from normal livers have a low mutational burden and limited diversity of signatures 8 , which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100–500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1–5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others—arising from exogenous exposures—were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease. Whole-genome sequencing of liver microdissections from five healthy individuals and nine with cirrhosis demonstrates the effects of liver disease on the genome, including increased rates of mutation, complex structural variation and different mutational signatures.

A mouse model of liver damage has identified a population of Lrg5 + liver stem cells that can generate hepatoctyes and bile ducts in vivo. Wake-up call for liver stem cells Hans Clevers and colleagues have identified a quiescent population of adult liver stem cells that can be 'woken up' by damage. In mice subject to liver damage, small cells expressing the Wnt target gene Lgr5 accumulate near the bile ducts. One of these cells was used to grow large numbers of bipotent stem cells in vitro . The stem cells were converted to functional hepatocytes in vitro , and when liver organoids were transplanted into a mouse model of tyrosinemia type I liver disease, islands of apparently normal hepatocytes appeared in the liver. Whether these hepatocytes are fully functional is not yet known, but the results are promising for regenerative approaches in the liver. The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon 1 , stomach 2 and hair follicles 3 . A three-dimensional culture system allows long-term clonal expansion of single Lgr5 + stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture 2 , 4 , 5 . A crucial component of the culture medium is the Wnt agonist RSPO1 6 , the recently discovered ligand of LGR5 7 , 8 . Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ + cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5 + cells generate hepatocytes and bile ducts in vivo . Single Lgr5 + cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah −/− mice. These findings indicate that previous observations concerning Lgr5 + stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology. Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along transcriptional gradients and suggest the presence of previously uncharacterized structures within liver tissue.